In the present paper, dsDNA separation has been studied in a silicon micropillar array column using ion-pair RP-HPLC (IP-RP-HPLC). The deep-etched (32.0 μm) silicon micropillar array was fabricated by advanced deep UV lithography and by a dedicated Bosch etch process and then sealed by anodic bonding to a Pyrex glass. The pillar surface was subsequently conditioned hydrophobic. Working in isocratic mode under nonretained conditions, van Deemter curves of dsDNA and coumarin were established to assess the performance of the micropillar array column, resulting in plate heights of only a few micrometers. Working in gradient mode, separations of dsDNA fragments were evaluated. The relevant gradient operation parameters were studied to understand their influence on dsDNA separations. The correlation between DNA length and retention was measured and theoretically described in a length range of 50-500 bp, promising for the determination of DNA of an unknown length. Finally, a separation example demonstrated the excellent separation power of on-chip IP-RP chromatography by achieving a large operation range of DNA length (10-300 bp) with a 5-bp difference among 11 dsDNA fragments.